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Aminopterin curative effect SNP locus genotype detection method and kit

A methotrexate and genotype technology, applied in biochemical equipment and methods, recombinant DNA technology, and microbial assay/examination, etc., can solve problems such as decreased curative effect, and achieve the effect of guiding clinical treatment selection

Inactive Publication Date: 2018-05-08
GUANGZHOU HEKANG MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The study found that MTHFR gene 1298C (rs1801131) in RA patients is associated with decreased curative effect and toxicity of MTX

Method used

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  • Aminopterin curative effect SNP locus genotype detection method and kit
  • Aminopterin curative effect SNP locus genotype detection method and kit
  • Aminopterin curative effect SNP locus genotype detection method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] A kit for detecting the genotype of the SNP site for methotrexate curative effect, the kit includes primers with the following sequences:

[0025]

[0026] The kit also includes a specific probe, and the sequence of the specific probe is as follows:

[0027]

[0028] The kit also includes a reporter probe, and the sequence of the reporter probe is as follows:

[0029]

Embodiment 2

[0031] 3 SNP sites were carried out in the same tube for PCR reaction. The reaction system had a total volume of 10 μl, including 2x qiagenHotstar MM 5ul, primer mix 1ul, DNA sample 2ul, and sterilized water 2ul.

[0032] The reaction was carried out on the ABI9700 PCR amplification instrument, the reaction conditions were 95°C, 15min, and 30 cycles of 94°C, 30 seconds, 60°C, 30 seconds, 72°C, 30 seconds; 72°C, 7 minutes, 4°C maintain.

[0033] Multiplex OLA reaction: Prepare 2xOLA master mix: 10x Taq Ligase buffer 2ul, Taq DNALigase (40,000U / ml) 0.25ul, wild-type probe mix (100nM each) 1ul, mutant probe mix (2.5uMeach) 2ul, deionized Water 4.75ul. Mix OLA master mix with the reaction product: 2xOLA master mix 10ul, amplified PCR product 5ul, sterilized deionized water 5ul. Pipette up and down to mix well, cover the reaction tube, and carry out the ligation reaction on the ABI9700 PCR amplification instrument. 96°C for 2min, 30 cycles of 94°C for 15s, 37°C for 1min; maintai...

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Abstract

The invention discloses an aminopterin curative effect SNP locus genotype detection method and kit, and relates to the fields of molecular biology and medicine. In the kit, aiming at 3 SNP loci havingaminopterin curative effect and toxic and side effect risks in human genome DNA, a specific primer and a wild / mutant probe are designed, hybridization reaction with MagPlex-TAG magnetic beads with aspecific reporting probe is performed, and detection is performed on a luminex 200 instrument through chromogenic reaction. A base type of each SNP locus is judged through reading and interpretation of signal values. The method and the kit have the beneficial effects that aiming at the 3 SNP loci related to the curative effect and toxic and side effect risks of aminopterin taken by patients with rheumatoid arthritis, the specific primer and the probe are designed, so that the type of the 3 SNP loci can be detected in a typing manner. The detection kit provided by the invention can detect the type of the 3 SNP loci in a human whole blood sample sensitively and quickly, can provide reliable experimental evidence for predicting the curative effect and toxic and side effect risks of aminopterin medication and guide clinical treatment selection.

Description

technical field [0001] The present invention relates to the fields of molecular biology and medicine. More specifically, the present invention relates to a method and a kit for detecting the SNP site genotype of the curative effect of methotrexate, by simultaneously detecting the curative effect of methotrexate and the risk of side effects The closely related single nucleotide polymorphism (SNP) genotypes of ABCB1 gene and MTHFR gene were used to evaluate the effectiveness and risk of side effects of methotrexate in rheumatoid arthritis and other diseases. Background technique [0002] Rheumatoid arthritis (RA) is a systemic autoimmune disease with arthritis as the main clinical manifestation, affecting 0.5%-1.0% of the population in the world, and the incidence rate in China is about 0.3%. Its basic pathological feature is vasculitis and synovitis. Synovial blood vessels in the joint proliferate and form pannus, leading to thickening of the synovium, secretion of various c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/106C12Q2600/156
Inventor 杜予和黄尚春
Owner GUANGZHOU HEKANG MEDICAL TECH CO LTD
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